We have recently characterized cDNA clones encoding PTH/PTHrP receptors from rat, human, opossum and murine cell lines and from rat and human kidney tissues. these clones provide the necessary tools to study the structural and functional features of these receptors. Current data are consistent with expression of the same receptor in bone and kidney. Northern blot analyses, however, show multiple mRNA species that suggest other receptors with related sequences. Additionally, other receptor subtypes that have limited (or no) sequence homology to the cloned cDNA also may be present. Therefore, the goals of Specific Aim 1 are to isolate other receptor subtypes by low-stringency hybridization and by expression cloning; and also to characterize the different receptor mRNAs seen on Northern blot analysis. The structural features underlying the capacity of the receptor to activate intracellular effectors are the subject of specific Aim 2. Using molecular biological and biochemical tools, G protein coupling domains of the PTH/PTHrP receptor will be characterized at the molecular level. The role of the cytoplasmic loops and the carboxy-terminal tail of the PTH/PTHrP receptor in G protein- coupling and effector-activation will be determined. Since the PTH receptor has the capability to activate more than one effector molecule, we will determine whether this occurs through one or multiple G proteins. Therefore, the goal of Specific Aim 3 is to determine which G protein and which effector molecule is activated by the PTH receptor. since the number of receptors is subject to tight regulation by PTH and PTHrP the molecular basis for receptor regulation will be studied in Specific Aim 4. Models of target cells, stably and constitutively expressing an epitope-tagged PTH/PTHrP receptor, will be used to study regulation of the receptor protein that is not due to regulation at the transcriptional level. Since phosphorylation of several G protein-coupled receptors plays an essential role in receptor regulation, phosphorylation of the PTH receptor by known protein kinases and potentially by PTH receptor- specific kinase will be assessed. The molecular determinants that underlie PTH receptor internalization, uncoupling from the G proteins, degradation and internalization will be mapped by both deletion and point mutation. These studies will provide insight into the molecular mechanisms that determine the functions of the PTH receptor in target cells. These studies will expand knowledge of the early events of PTH and PTHrP interactions with common receptors, and potentially or receptors that might bind each protein specifically. Characterizing the specificity of PTH/PTHrP receptor coupling to G proteins and its molecular basis, and the mechanisms that regulate these receptors will both define these properties of these receptor(s)'s, and will serve as models for other receptors in this novel of G protein-linked receptor family. Increased understanding of PTH and PTHrP receptor functions will provide insights into their physiological roles, and together with the other projects in this Program, will lead to more rational drug design for human disease, particularly bone-loss states such as osteoporosis.